An altered expression of genes involved in the

EXPERIMENTAL AND THERAPEUTIC MEDICINE 5: 1239-1343, 2013

An altered expression of genes involved in the regulation of ion channels in atrial myocytes is correlated with the risk of atrial fibrillation in patients with heart failure MEI GAO, JIANGRONG WANG, ZHONGSU WANG, YONG ZHANG, HUI SUN, XINXING XIE and YINGLONG HOU Department of Cardiology, Shandong Provincial Qianfoshan Hospital, Shandong University, Jinan, P.R. China Received December 3, 2012; Accepted January 22, 2013 DOI: 10.3892/etm.2013.949 Abstract. The aim of this study was to investigate the correlation between the altered expression of genes involved in the regulation of ion channels in atrial myocytes and the risk of atrial fibrillation (AF) in patients with heart failure (HF). Right atrial appendages were obtained from 18 HF patients and 18 patients with normal cardiac functions who had undergone surgery. The mRNA expression levels of Kv4.3α, KvLQT1, Kv1.5, L-Caα1c and NCX were measured by reverse transcription-PCR (RT-PCR). Protein expression levels were also detected by western blotting. In comparison with the control group exhibiting normal cardiac functions, the mRNA and protein expression levels of Kv4.3α, KvLQT1 and L-Caα1c were significantly reduced in HF patients. By contrast, the mRNA and protein expression levels of NCX were significantly increased in HF patients compared with the control group (P1.8 and thereafter stored at -20˚C for detection. RT-PCR technology was applied for reverse transcription and cDNA fragment amplification. The first-strand cDNA was synthesized using AMV reverse transcriptase. Total RNA was reverse transcribed in a final volume of 10 µl, containing the following: 2 µl MgCl 2 (25 mM); 1 µl 10X RT buffer; 3.75 µl RNase free dH2O; 1 µl dNTP mixture (10 mM); 0.25 µl RNase inhibitor; 0.5 µl AMV reverse transcriptase; 0.5 µl oligo dT-adaptor primer; 1 µl RNA. The reverse transcription was then conducted as follows: the reac-

tion mixture was incubated at 30˚C for 10 min, annealed at 42˚C for 30 min, followed by incubation at 99˚C for 5 min and 5˚C for 5 min. Following denaturation at 94˚C for 2 min, the samples were subjected to 30 cycles of denaturation at 94˚C for 30 sec, annealing for 30 sec and extension at 72˚C for 50 sec. Then, 35 cycle PCR amplification was used with a 5-min extension time (reaction solution 2). The 10 µl of amplified product was electrophoresed in 1.5% agarose gel containing ethidium bromide, examined and photographed under a UV transilluminator. The intensity of each band was quantified using image analysis software (TINA version 2.10, Raytest, Straubenhardt, Germany) and the expression levels were calculated by measuring the OD of the target gene and normalized to that of the amplified GAPDH. Western blot analysis. All relevant proteins were harvested from tissue, separated by 10% SDS/PAGE and then subjected to immunoblot analyses. The primary antibodies against Kv4.3 α, KvLQT1, Kv1.5, L-Ca α1c, NCX and actin were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA; anti-Kv4.3α, cat# sc-11686, 1:200; anti-KvLQT1, cat# sc-365186, 1:200; anti-Kv1.5, cat# sc-377110, 1:200; antiL-Caα1c, cat# sc-166069, 1:200; anti-NCX, cat# sc-32881, 1:200; anti-actin, cat# sc-130301, 1:10,000). Secondary antibodies used in this study were donkey anti-goat IgG-HRP (cat# sc-2020, 1:5,000, Santa Cruz Biotechnology, Inc.), goat anti-rabbit IgG-HRP (cat# sc-2004, 1:5,000, Santa Cruz Biotechnology, Inc.) and goat anti-mouse IgG-HRP (cat# sc-2005, 1:10,000, Santa Cruz Biotechnology, Inc.). Bound antibodies were detected using the ECL system (Pierce Biotechnology, Inc., Rockford, IL, USA). The immunoblot experiments were repeated at least 3 times. The mean normalized OD of detected Kv4.3α, KvLQT1, Kv1.5, L-Caα1c or NCX protein bands relative to the OD of the actin band from the same individual was calculated, respectively. Statistical analysis. Concise Statistics 2000 was used to perform the statistical analyses. All numerical values are expressed as mean ± SD. The t-test was performed for comparison of the experimental group and the control group. P